Knecht W, Henseling J, Löffler M
School of Medicine, Institute for Physiological Chemistry, Phillipps University Marburg, Germany.
Chem Biol Interact. 2000 Jan 3;124(1):61-76. doi: 10.1016/s0009-2797(99)00144-1.
Mitochondrially-bound dihydroorotate dehydrogenase (EC 1.3.99.11) catalyzes the fourth sequential step in the de novo synthesis of uridine monophosphate. The enzyme has been identified as or surmised to be the pharmacological target for isoxazol, triazine, cinchoninic acid and (naphtho)quinone derivatives, which exerted antiproliferative, immunosuppressive, and antiparasitic effects. Despite this broad spectrum of biological and clinical relevance, there have been no comparative studies on drug-dihydroorotate dehydrogenase interactions. Here, we describe a study of the inhibition of the purified recombinant human and rat dihydroorotate dehydrogenase by ten compounds. 1,4-Naphthoquinone, 5,8-hydroxy-naphthoquinone and the natural compounds juglon, plumbagin and polyporic acid (quinone derivative) were found to function as alternative electron acceptors with 10-30% of control enzyme activity. The human and rat enzyme activity was decreased by 50% by the natural compound lawsone ( > 500 and 49 microM, respectively) and by the derivatives dichloroally-lawsone (67 and 10 nM), lapachol (618 and 61 nM) and atovaquone (15 microM and 698 nM). With respect to the quinone co-substrate of the dihydroorotate dehydrogenase, atovaquone (Kic = 2.7 microM) and dichloroally-lawsone (Kic = 9.8 nM) were shown to be competitive inhibitors of human dihydroorotate dehydrogenase. Atovaquone (Kic = 60 nM) was also acompetitive inhibitor of the rat enzyme. Dichloroally]-lawsone was found to be a time-dependent inhibitor of the rat enzyme, with the lowest inhibition constant (Ki* = 0.77 nM) determined so far for mammalian dihydroorotate dehydrogenases. Another inhibitor, brequinar was previously reported to be a slow-binding inhibitor of the human dihydroorotate dehydrogenase [W. Knecht, M. Loffler, Species-related inhibition of human and rat dihyroorotate dehydrogenase by immunosuppressive isoxazol and cinchoninic acid derivatives, Biochem. Pharmacol. 56 (1998) 1259-1264]. The slow binding features of this potent inhibitor (Ki* = 1.8 nM) with the human enzyme, were verified and seen to be one of the reasons for the narrow therapeutic window (efficacy versus toxicity) reported from clinical trials on its antiproliferative and immunosuppressive action. With respect to the substrate dihydroorotate, atovaquone was an uncompetitive inhibitor of human dihydroorotate dehydrogenase (Kiu = 11.6 microM) and a non-competitive inhibitor of the rat enzyme (Kiu = 905/ Kic = 1,012 nM). 1.5 mM polyporic acid, a natural quinone from fungi, influenced the activity of the human enzyme only slightly; the activity of the rat enzyme was decreased by 30%.
线粒体结合的二氢乳清酸脱氢酶（EC 1.3.99.11）催化尿苷单磷酸从头合成中的第四步连续反应。该酶已被确定或推测为异恶唑、三嗪、喹啉酸和（萘）醌衍生物的药理学靶点，这些衍生物具有抗增殖、免疫抑制和抗寄生虫作用。尽管具有如此广泛的生物学和临床相关性，但尚未有关于药物 - 二氢乳清酸脱氢酶相互作用的比较研究。在此，我们描述了一项关于十种化合物对纯化的重组人及大鼠二氢乳清酸脱氢酶抑制作用的研究。发现1,4 - 萘醌、5,8 - 羟基萘醌以及天然化合物胡桃醌、白花丹素和多孔酸（醌衍生物）可作为替代电子受体，酶活性为对照的10 - 30%。天然化合物胡桃酮（分别大于500和49 microM）以及衍生物二氯烯丙基 - 胡桃酮（67和10 nM）、拉帕醇（618和61 nM）和阿托伐醌（15 microM和698 nM）可使人和大鼠的酶活性降低50%。就二氢乳清酸脱氢酶的醌辅底物而言，阿托伐醌（Kic = 2.7 microM）和二氯烯丙基 - 胡桃酮（Kic = 9.8 nM）被证明是人类二氢乳清酸脱氢酶的竞争性抑制剂。阿托伐醌（Kic = 60 nM）也是大鼠酶的竞争性抑制剂。二氯烯丙基 - 胡桃酮被发现是大鼠酶的时间依赖性抑制剂，其抑制常数（Ki* = 0.77 nM）是迄今为止测定的哺乳动物二氢乳清酸脱氢酶中最低的。另一种抑制剂布喹那先前被报道为人类二氢乳清酸脱氢酶的慢结合抑制剂[W. Knecht, M. Loffler, 免疫抑制异恶唑和喹啉酸衍生物对人和大鼠二氢乳清酸脱氢酶的种属相关抑制作用，生物化学与药物学，56 (1998) 1259 - 1264]。这种强效抑制剂（Ki* = 1.8 nM）与人类酶的慢结合特性得到了验证，并且被认为是其抗增殖和免疫抑制作用的临床试验中报道的治疗窗狭窄（疗效与毒性）的原因之一。就底物二氢乳清酸而言，阿托伐醌是人类二氢乳清酸脱氢酶的非竞争性抑制剂（Kiu = 11.6 microM），是大鼠酶的非竞争性抑制剂（Kiu = 905 / Kic = 1,012 nM）。1.5 mM多孔酸，一种来自真菌的天然醌，对人类酶活性的影响很小；大鼠酶的活性降低了30%。
